The patch clamp technique, developed in late 1970s, started a new period of experimental cardiac electrophysiology enabling measurement of ionic currents on isolated cardiomyocytes down to the level of single channels. Since that time, the technique has been substantially improved by development of several upgraded modifications providing so far unavailable data (e.g. action potential clamp, dynamic clamp, high-resolution scanning patch clamp), or facilitating the patch clamp technique by increasing its efficiency (planar patch clamp, automated patch clamp). The current review summarizes the leading new patch clamp based techniques used in cardiac cellular electrophysiology, their principles and prominent related papers.

• MeSH
• akční potenciály fyziologie   MeSH
• design vybavení trendy   MeSH
• gating iontového kanálu fyziologie   MeSH
• iontové kanály metabolismus   MeSH
• lidé MeSH
• membránové potenciály fyziologie   MeSH
• metoda terčíkového zámku přístrojové vybavení   trendy   MeSH
• mikroelektrody trendy   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• iontové kanály MeSH

Patch clamp method developed more than 30 years ago is widely used for investigation of cellular excitability manifested as transmembrane ionic current and/or generation of action potentials. This technique could be applied to measurement of ionic currents flowing through individual (single) ion channels or through the whole assembly of ion channels expressed in the whole cell. Whole cell configuration is more common for measurement of ion currents and the only one enabling measurement of action potentials. This method allows detailed analysis of mechanisms and structural determinants of voltage-dependent gating of ion channels as well as regulation of channel activity by intracellular signaling pathways and pharmacological agents.

• MeSH
• akční potenciály MeSH
• biologické modely MeSH
• buněčná membrána metabolismus   MeSH
• gating iontového kanálu MeSH
• iontové kanály metabolismus   MeSH
• lidé MeSH
• membránové potenciály MeSH
• metoda terčíkového zámku * MeSH
• vápníková signalizace MeSH
• vápníkové kanály - typ T metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• iontové kanály MeSH
• vápníkové kanály - typ T MeSH

We have developed an improved technique for fast cooling and heating of solutions superfusing isolated cells under patch-clamp or calcium imaging conditions. The system meets the requirements for studying temperature dependency of all kinds of ion channels, in particular temperature-gated ion channels. It allows the application of temperature changes within a range of 5-60 degrees C at maximum rates of -40 degrees C/s to 60 degrees C/s. Barrels filled with different solutions are connected to a manifold consisting of seven silica capillaries (320 microm inner diameter, i.d.). A common outlet consists of a glass capillary through which the solutions are applied onto the cell surface. The upper part of this capillary is embedded in a temperature exchanger driven by a miniature Peltier device which preconditions the temperature of the passing solution. The lower part of the capillary carries an insulated copper wire, densely coiled over a length of 7 mm, and connected to a dc current source for resistive heating. The Peltier device and the heating element are electrically connected to the headstage probe which is fixed on to a micromanipulator for positioning of the manifold. The temperature of the flowing solution is measured by a miniature thermocouple inserted into the common outlet capillary near to its orifice which is placed at a distance of less than 100 microm from the surface of the examined cell. The temperature is either manually controlled by voltage commands or adjusted via the digital-to-analog converter of a conventional data acquisition interface. Examples are given of using the device in patch-clamp studies on heterologously expressed TRPV1, TRPM8, and on cultured rat sensory neurons.

• MeSH
• akční potenciály fyziologie   MeSH
• analýza selhání vybavení MeSH
• buněčné kultury přístrojové vybavení   metody   MeSH
• design vybavení MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• lidé MeSH
• membránové potenciály fyziologie   MeSH
• metoda terčíkového zámku přístrojové vybavení   metody   MeSH
• neurony aferentní fyziologie   MeSH
• nízká teplota * MeSH
• perfuze přístrojové vybavení   MeSH
• prostředí kontrolované MeSH
• vysoká teplota * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH

• MeSH
• biologický transport MeSH
• buněčná membrána metabolismus   MeSH
• draslík metabolismus   MeSH
• draslíkové kanály metabolismus   MeSH
• membránové proteiny genetika   metabolismus   MeSH
• metoda terčíkového zámku MeSH
• proteiny přenášející kationty * MeSH
• Schizosaccharomyces pombe - proteiny * MeSH
• Schizosaccharomyces genetika   metabolismus   MeSH
• transportní proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• draslík MeSH
• draslíkové kanály MeSH
• membránové proteiny MeSH
• proteiny přenášející kationty * MeSH
• Schizosaccharomyces pombe - proteiny * MeSH
• transportní proteiny MeSH
• trk1 protein, S pombe MeSH Prohlížeč

• MeSH
• buněčná membrána fyziologie   MeSH
• elektrofyziologie přístrojové vybavení   MeSH
• iontové kanály fyziologie   MeSH
• kuřecí embryo MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• iontové kanály MeSH

The ankyrin transient receptor potential channel TRPA1 is a polymodal sensor for noxious stimuli, and hence a promising target for treating chronic pain. This tetrameric six-transmembrane segment (S1-S6) channel can be activated by various pungent chemicals, such as allyl isothiocyanate or cinnamaldehyde, but also by intracellular Ca(2+) or depolarizing voltages. Within the S4-S5 linker of human TRPA1, a gain-of-function mutation, N855S, was recently found to underlie familial episodic pain syndrome, manifested by bouts of severe upper body pain, triggered by physical stress, fasting, or cold. To clarify the structural basis for this channelopathy, we derive a structural model of TRPA1 by combining homology modeling, molecular dynamics simulations, point mutagenesis and electrophysiology. In the vicinity of N855, the model reveals inter-subunit salt bridges between E854 and K868. Using the heterologous expression of recombinant wild-type and mutant TRPA1 channels in HEK293T cells, we indeed found that the charge-reversal mutants E854R and K868E exhibited dramatically reduced responses to chemical and voltage stimuli, whereas the charge-swapping mutation E854R/K868E substantially rescued their functionalities. Moreover, mutation analysis of highly conserved charged residues within the S4-S5 region revealed a gain-of-function phenotype for R852E with an increased basal channel activity, a loss of Ca(2+)-induced potentiation and an accelerated Ca(2+)-dependent inactivation. Based on the model and on a comparison with the recently revealed atomic-level structure of the related channel TRPV1, we propose that inter-subunit salt bridges between adjacent S4-S5 regions are crucial for stabilizing the conformations associated with chemically and voltage-induced gating of the TRPA1 ion channel.

• Klíčová slova
• Ankyrin receptor subtype 1, Homology modeling, Molecular dynamics, Mutagenesis, S4–S5-linker, Transient receptor potential,
• MeSH
• asparagin genetika   MeSH
• elektrická stimulace MeSH
• gating iontového kanálu účinky léků   fyziologie   MeSH
• HEK293 buňky MeSH
• isothiokyanatany farmakologie   MeSH
• kationtové kanály TRP chemie   genetika   metabolismus   MeSH
• kationtový kanál TRPA1 MeSH
• lidé MeSH
• membránové potenciály genetika   MeSH
• metoda terčíkového zámku MeSH
• molekulární modely * MeSH
• mutace genetika   MeSH
• mutageneze MeSH
• proteiny nervové tkáně chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• serin genetika   MeSH
• terciární struktura proteinů MeSH
• transfekce MeSH
• vápník metabolismus   MeSH
• vápníkové kanály chemie   genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 2,3,4-tri-O-acetylarabinopyranosyl isothiocyanate MeSH Prohlížeč
• asparagin MeSH
• isothiokyanatany MeSH
• kationtové kanály TRP MeSH
• kationtový kanál TRPA1 MeSH
• proteiny nervové tkáně MeSH
• serin MeSH
• TRPA1 protein, human MeSH Prohlížeč
• vápník MeSH
• vápníkové kanály MeSH

Rat melanotrophs express several types of voltage-gated and ligand-gated calcium channels, although mechanisms involved in the maintenance of the resting intracellular Ca2+ concentration ([Ca2+]i) remain unknown. We analyzed mechanisms regulating resting [Ca2+]i in dissociated rat melanotrophs by Ca2+-imaging and patch-clamp techniques. Treatment with antagonists of L-type, but not N- or P/Q-type voltage-gated Ca2+ channels (VGCCs) as well as removal of extracellular Ca2+ resulted in a rapid and reversible decrease in [Ca2+]i, indicating constitutive Ca2+ influx through L-type VGCCs. Reduction of extracellular Na+ concentration (replacement with NMDG+) similarly decreased resting [Ca2+]i. When cells were champed at -80 mV, decrease in the extracellular Na+ resulted in a positive shift of the holding current. In cell-attached voltage-clamp and whole-cell current-clamp configurations, the reduction of extracellular Na+ caused hyperpolarisation. The holding current shifted in negative direction when extracellular K+ concentration was increased from 5 mM to 50 mM in the presence of K+ channel blockers, Ba2+ and TEA, indicating cation nature of persistent conductance. RT-PCR analyses of pars intermedia tissues detected mRNAs of TRPV1, TRPV4, TRPC6, and TRPM3-5. The TRPV channel blocker, ruthenium red, shifted the holding current in positive direction, and significantly decreased the resting [Ca2+]i. These results indicate operation of a constitutive cation conductance sensitive to ruthenium red, which regulates resting membrane potential and [Ca2+]i in rat melanotrophs.

• Klíčová slova
• Ca(2+) homeostasis, Cation conductance, Patch-clamp, Ca(2+) imaging, ruthenium red, Rat melanotrophs, Voltage-gated calcium channels,
• MeSH
• kationtové kanály TRPV antagonisté a inhibitory   metabolismus   MeSH
• krysa rodu Rattus MeSH
• melanotropní buňky metabolismus   MeSH
• metoda terčíkového zámku MeSH
• potkani Wistar MeSH
• rutheniová červeň farmakologie   MeSH
• sodík metabolismus   MeSH
• vápník metabolismus   MeSH
• vápníkové kanály - typ L metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kationtové kanály TRPV MeSH
• rutheniová červeň MeSH
• sodík MeSH
• vápník MeSH
• vápníkové kanály - typ L MeSH

N-methyl-D-aspartate receptor (NMDAR) hypofunction has been implicated in several neurodevelopmental disorders. NMDAR function can be augmented by positive allosteric modulators, including endogenous compounds, such as cholesterol and neurosteroid pregnenolone sulfate (PES). Here we report that PES accesses the receptor via the membrane, and its binding site is different from that of cholesterol. Alanine mutagenesis has identified residues that disrupt the steroid potentiating effect at the rat GluN1 (G638; I642) and GluN2B (W559; M562; Y823; M824) subunit. Molecular dynamics simulation indicates that, in the absence of PES, the GluN2B M1 helix residue W559 interacts with the M4 helix residue M824. In the presence of PES, the M1 and M4 helices of agonist-activated receptor rearrange, forming a tighter interaction with the GluN1 M3 helix residues G638 and I642. This stabilizes the open-state position of the GluN1 M3 helices. Together, our data identify a likely binding site for the NMDAR-positive allosteric modulator PES and describe a novel molecular mechanism by which NMDAR activity can be augmented.SIGNIFICANCE STATEMENT There is considerable interest in drugs that enhance NMDAR function and could compensate for receptor hypofunction associated with certain neuropsychiatric disorders. Positive allosteric modulators of NMDARs include an endogenous neurosteroid pregnenolone sulfate (PES), but the binding site of PES on the NMDAR and the molecular mechanism of potentiation are unknown. We use patch-clamp electrophysiology in combination with mutagenesis and in silico modeling to describe the interaction of PES with the NMDAR. Our data indicate that PES binds to the transmembrane domain of the receptor at a discrete group of residues at the GluN2B membrane helices M1 and M4 and the GluN1 helix M3, and that PES potentiates NMDAR function by stabilizing the open-state position of the GluN1 M3 helices.

• Klíčová slova
• glutamate receptors, neurosteroids, patch clamp, structure,
• MeSH
• alanin genetika   MeSH
• buněčná membrána účinky léků   MeSH
• cholesterol metabolismus   MeSH
• elektrofyziologické jevy MeSH
• HEK293 buňky MeSH
• konformace proteinů MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• metoda terčíkového zámku MeSH
• pregnenolon farmakologie   MeSH
• receptory N-methyl-D-aspartátu účinky léků   MeSH
• simulace molekulární dynamiky MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alanin MeSH
• cholesterol MeSH
• NMDA receptor A1 MeSH Prohlížeč
• NR2B NMDA receptor MeSH Prohlížeč
• pregnenolon MeSH
• pregnenolone sulfate MeSH Prohlížeč
• receptory N-methyl-D-aspartátu MeSH

A characteristic of mast cells is the degranulation in response to various stimuli. Here we have investigated the effects of various physical stimuli in the human mast-cell line HMC-1. We have shown that HMC-1 express the transient receptor potential channels TRPV1, TRPV2 and TRPV4. In the whole-cell patch-clamp configuration, increasing mechanical stress applied to the mast cell by hydrostatic pressure (-30 to -90 cm H(2)O applied via the patch pipette) induced a current that could be inhibited by 10 microM of ruthenium red. This current was also inhibited by 20 microM SKF96365, an inhibitor that is among TRPV channels specific for the TRPV2. A characteristic of TRPV2 is its activation by high noxious temperature; temperatures exceeding 50 °C induced a similar ruthenium-red-sensitive current. As another physical stimulus, we applied laser light of 640 nm. Here we have shown for the first time that the application of light (at 48 mW for 20 min) induced an SKF96365-sensitive current. All three physical stimuli that led to activation of SKF96365-sensitive current also induced pronounced degranulation in the mast cells, which could be blocked by ruthenium red or SKF96365. The results suggest that TRPV2 is activated by the three different types of physical stimuli. Activation of TRPV2 allows Ca(2+) ions to enter the cell, which in turn will induce degranulation. We, therefore, suggest that TRPV2 plays a key role in mast-cell degranulation in response to mechanical, heat and red laser-light stimulation.

• MeSH
• buněčné linie MeSH
• degranulace buněk MeSH
• exocytóza MeSH
• kationtové kanály TRPV metabolismus   MeSH
• lidé MeSH
• mastocyty fyziologie   MeSH
• metoda terčíkového zámku MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kationtové kanály TRPV MeSH
• TRPV2 protein, human MeSH Prohlížeč

Alterations in ion channel expression and function known as "electrical remodeling" contribute to the development of hypertrophy and to the emergence of arrhythmias and sudden cardiac death. However, comparing current density values - an electrophysiological parameter commonly utilized to assess ion channel function - between normal and hypertrophied cells may be flawed when current amplitude does not scale with cell size. Even more, common routines to study equally sized cells or to discard measurements when large currents do not allow proper voltage-clamp control may introduce a selection bias and thereby confound direct comparison. To test a possible dependence of current density on cell size and shape, we employed whole-cell patch-clamp recording of voltage-gated sodium and calcium currents in Langendorff-isolated ventricular cardiomyocytes and Purkinje myocytes, as well as in cardiomyocytes derived from trans-aortic constriction operated mice. Here, we describe a distinct inverse relationship between voltage-gated sodium and calcium current densities and cell capacitance both in normal and hypertrophied cells. This inverse relationship was well fit by an exponential function and may be due to physiological adaptations that do not scale proportionally with cell size or may be explained by a selection bias. Our study emphasizes the need to consider cell size bias when comparing current densities in cardiomyocytes of different sizes, particularly in hypertrophic cells. Conventional comparisons based solely on mean current density may be inadequate for groups with unequal cell size or non-proportional current amplitude and cell size scaling.

• Klíčová slova
• L-type calcium and sodium current density, Ventricular and Purkinje myocyte, bias, cell capacitance, cell size, trans-aortic constriction induced hypertrophy,
• MeSH
• kardiomegalie * metabolismus   patologie   MeSH
• kardiomyocyty * metabolismus   patologie   MeSH
• metoda terčíkového zámku MeSH
• myši MeSH
• velikost buňky * MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH